Revolutionizing Stress Monitoring: Multimodal Responses and Segmented Populations in Biofilms Unveiled by a Three-Colour Stress Biosensor

A new biosensor system, dubbed RGB-S Reporter, enables the simultaneous monitoring of three different stress responses in E. coli, revealing heterogeneous responses in planktonic single cells and live biofilms
Published in Microbiology
Revolutionizing Stress Monitoring: Multimodal Responses and Segmented Populations in Biofilms Unveiled by a Three-Colour Stress Biosensor

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Stress Responses and the Quest for Bioreporters

Microbial life is a constant interplay of responses to various environmental stressors. From changes in temperature to exposure to toxic substances, microorganisms have evolved intricate mechanisms to safeguard their survival. Unveiling these stress response pathways is not only crucial for basic scientific understanding but also holds significant potential for diverse applications in technology and medicine. The advent of bioreporters, those molecular sensors capable of revealing a cell's internal state, has been a game-changer. These tools provide insights into cellular behavior under various conditions, paving the way for applications ranging from ecological studies to toxic substance screening. Bioreporters act as molecular "feelers" and enable researchers to observe changes in gene expression by emitting signals that correlate with a cell's physiological state.

However, traditional bioreporters to probe cell responses to stress have often been laborious, invasive, and limited in scope. Most bioreporters employ mono or dual-color systems, limiting their ability to capture complex responses involving multiple stress pathways. Furthermore, traditional bioreporter systems often necessitate biochemical assays or rely on in vivo reactions to generate visible signals, making real-time monitoring challenging. As the demand for more versatile, accurate, and noninvasive tools grew, we saw an opportunity to bridge this gap.

Rising to the Challenge: The RGB-S Biosensor for Multiplexed Stress Sensing

Our vision was simple yet transformative – to develop a bioreporter system that could capture multiple stress responses simultaneously, independently, in real time, with high precision and minimal interference. By leveraging the potential of fluorescence-based reporters and stress-specific expression promoters, we developed the RGB-S reporter, a genetically-encoded three-color fluorescent biosensor (Fig. 1). The new system harnesses the power of three distinct fluorescent proteins—red, green, and blue— to precisely report three major cellular responses to physiological stress (RpoS), genotoxicity (SOS) and cytotoxicity (RpoH). The system was designed to overcome the limitations of existing technologies making it easy to use, reliable, and capable of generating detectable signals even at very low stressor concentrations. Among the standout features of the RGB-S reporter is its rapid response time, delivering insights about stress state in a matter of minutes. The non-overlapping fluorescence spectra of the three colors along with the carefully picked promoters not only enabled simultaneous observation of the responses with high specificity but also allowed us to track changes in stress levels over time. Additionally, the stability of the biosensor's plasmid even at stressful conditions ensures consistent and reliable performance over time.

Fig. 1: RGB-S reporter system for monitoring bacterial starvation, genotoxicity and cytotoxicity.

Unmasking Intriguing Discoveries

The RGB-S biosensor's debut brought to light an array of discoveries that previously remained obscured. In the past, stress response studies often focused on isolated pathways, missing the complex interplay between different stressors. With the RGB-S biosensor, our experiments demonstrated that cells can exhibit both monomodal and multimodal stress responses within the same cell. This rich diversity of responses offered valuable insights into the intricate ways microorganisms navigate their survival, indicating a remarkable degree of adaptability. Furthermore, this novel tool in combination with microfluidic cultivation and confocal fluorescence microscopy imaging has enabled the observation of spatiotemporal (i.e. in space and time) stress dynamics within microbial biofilms. The intricate world of stress in biofilms was unveiled with surprising insights, revealing stratified/localized subpopulations of bacteria with varying stress responses, as if each layer played a unique role in the survival of the community (Fig. 2).

Fig. 2: Stratified organization of stress responses in biofilm matrix. RFP, GFP, and BFP indicate physiological stress, genotoxicity and cytotoxicity, respectively. Scale bars are 100 µm.

From Lab Bench to Broader Impact

The implications of the RGB-S biosensor extend beyond the laboratory bench. On the one hand, we can now delve deeper into the mechanisms of stress responses, unraveling the intricate orchestration of pathways that underpin microbial adaptability. The ability to capture comprehensive stress data, coupled with the unprecedented insights into biofilm dynamics, promises to unlock new avenues of research. On the applied front, the biosensor's versatility opens doors to identifying toxic substances with respect to environmental monitoring. By deciphering stressful conditions for microorganisms under bioproduction settings, industries can refine their practices, leading to more efficient biotechnological processes and quicker assessments of stress-induced cell viability and productivity.

In conclusion, the journey from mono or dual-color bioreporters to the RGB-S reporter represents a leap forward in microbial stress monitoring technology. This innovation holds immense promise for our continued exploration of the microbial world and its intricate interactions with its surroundings. The RGB-S biosensor's ability to simultaneously track multiple stress pathways and its capacity to unveil the hidden dynamics of microbial biofilms positions it as a transformative tool in both basic and applied research, illuminating a path towards a deeper understanding of the intricate world of microbial stress responses, promising a future filled with innovative discoveries and applications.

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Go to the profile of Sudarsan Mugunthan
6 months ago

Nice work. Please find my work on biofilms.

Go to the profile of Kersten Rabe
about 2 months ago

Thanks a lot for highlighting your research as well! Intersting work!

Go to the profile of Kersten Rabe
about 2 months ago

The plasmid is now available at addgene:

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